Streptococcus pneumoniae remains a major cause of morbidity and mortality worldwide. Two components of pneumococci are thought to be responsible for much of the deleterious inflammatory hot response, the thiol-activated secreted toxin pneumolysin and the peptidoglycan component of the ell wall. Both of these molecules have been shown to cause inflammatory changes in vitro studies in epithelial and neuronal cells, as well as activate monocytes and macrophages of the inactive immune system. Preliminary work in our laboratory supports the hypothesis that pneumolysin activates the innate arm of the immune system through a transmembrane receptor, Toll-like receptor 4 (TLR4), in synergy with peptidoglycan (a TLR2 ligand) and/or whole pneumococci. In addition, we have some preliminary data to suggest that the activation of the innate immune response by pneumolysin may greatly augment acquired immunity to pneumococcus as well. The scientific goals of this project are divided in two phases. In the first phase, the PI will study the TLR- dependent signaling pathway of pneumolysin, alone and in combination with peptidoglycan or whole pneumococci. Studies will be done to test the hypothesis that pneumolysin binds directly to TLR4. This phase will also include an intense didactic component, to complement the work in the laboratory. In the second phase, the PI will study the role of this pathway in the modulation of disease and acquired immunity to pneumococci. This phase of the project will test the hypothesis that TLR4-mediated cellular activation by pneumolysin plays an important role in the pathophysiology of pneumococcal disease. Pneumococci carrying mutations in the pneumolysin gene, with diminished or abrogated TLR4-signaling capability, will be studied both in vitro (in macrophages) and in vivo (in murine models of colonization and disease). The possible role of TLR4-mediated response to pneumolysin in the development of acquired immunity will be studied in the final aim of the proposal. The establishment of a link between the innate and the acquired arm of the immune response to pneumococcus may provide a better understanding of the mechanisms by which immunity to pneumococcus is acquired.